In Vitro and in Vivo Efficacy of Different Ointment Formulations Containing Centaurium erythraea Rafn. Aerial Extract

Abstract

Background: Centaurium erythraea Rafn. (C. erythraea) is a medicinal plant traditionally used in European folk medicine for the treatment of wounds, skin inflammations, and other dermatological conditions, in addition to its well-documented systemic antioxidant and anti-inflammatory effects. However, its topical applications remain insufficiently investigated, particularly using plant material collected from Romania. The purpose of this study was to prepare different ointment formulations containing C. erythraea Rafn. extract obtained from the aerial parts of the plant, using various excipients, and to evaluate their in vitro and in vivo efficacy.

Methods: The phytochemical profile of C. erythraea extract was characterized using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The lyophilized extract was pre-dissolved in different solubilizing agents—Transcutol® P (diethylene glycol monoethyl ether), Capryol® 90 (propylene glycol monocaprylate), or a combination of both—and then incorporated into five ointment formulations. Texture analysis and an in vitro membrane diffusion study were performed. The antioxidant capacity of the formulations was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP), and total phenolic content (TPC) assays. Anti-inflammatory activity was evaluated in vitro using tumor necrosis factor-alpha (TNF-α)-induced interleukin-1 beta (IL-1β) production in human keratinocyte (HaCaT) cells, and in vivo using a carrageenan-induced rat paw edema model.

Results: LC–MS/MS identified 18 polyphenolic compounds, with hyperoside (3.78 ± 0.05 µg/mL), protocatechuic acid (1.13 ± 0.06 µg/mL), chlorogenic acid (1.07 ± 0.06 µg/mL), and quercetin (0.53 ± 0.03 µg/mL) as the principal constituents. The formulation containing both Transcutol^® P and Capryol^® 90 exhibited the most pronounced antioxidant activity (65% DPPH inhibition; 69.71 ± 0.83 mg gallic acid equivalent/mL) and significantly reduced IL-1β levels by 45.7% compared to the inflamed control. In vivo, this formulation showed comparable anti-edematous effects to a methylprednisolone ointment. Furthermore, it demonstrated the highest skin permeation efficiency, with a quercetin diffusion coefficient of 35.12 × 10⁻⁵ cm²/min.

Conclusions: These findings highlight the therapeutic potential of C. erythraea extract from aerial parts in topical formulations and underscore the enhancing role of Transcutol® P and Capryol® 90 in improving both the pharmacodynamic and pharmacokinetic properties of bioactive compounds.

Introduction

Herbs hold significant importance in human health due to their wide range of therapeutic effects [1]. Over the past thirty years, the global use of herbal medicines and supplements has risen dramatically, with more than 80% of people now depending on them for their primary healthcare needs [2]. As the demand for alternative therapies grows, it is increasingly essential to critically assess their safety and efficacy. Comprehensive scientific investigation is crucial to ensuring that these compounds offer therapeutic benefits while minimizing potential risks, facilitating their informed and responsible integration into healthcare practices [3]. Centaurium erythraea Rafn. (C. erythraea), the plant with a thousand medicinal properties according to Hungarian folk medicine, is an important healing plant from Gentianaceae family, which is used traditionally in the folk medicine as a digestive, stomachic, tonic, depurative, sedative, and antipyretic herb [4,5]. C. erythraea is also one of the potential drugs to treat asthma, eczema, jaundice, fever, intestinal parasites, rheumatism, hypertension, gastrointestinal muscle spasms, and edema and to stimulate the activity of the liver and gallbladder [6,7]. C. erythraea demonstrated significant antidiabetic properties by enhancing insulin sensitivity and lowering blood glucose levels, making it a promising natural treatment for diabetes [8,9]. The aerial parts of C. erythraea have also shown anti-inflammatory and antioxidant effects attributed to xantones and secoiridoid glycosides, namely gentiopicroside from the plant [10,11,12,13].

Antioxidants in skin formulations help protect against oxidative stress by neutralizing reactive oxygen species. This reduces free radical–induced cellular damage, supporting skin integrity and delaying visible signs of aging [14,15]. C. erythraea exhibits potent antioxidant activity attributable to its high concentration of polyphenolic compounds. These bioactive constituents contribute to the reduction in oxidative stress and modulation of inflammatory pathways, promoting cytoprotective effects within skin tissue [4,16].

Inflammation is a complex, well-regulated process the body uses to respond to injury or infection, characterized by pain, redness, heat, swelling, and sometimes loss of function [17,18]. This response includes recognition of danger signals, activation of pathways, and recruitment of immune cells [18,19]. While generally beneficial for tissue repair, persistent or dysregulated inflammation can become chronic and harmful, leading to permanent tissue damage [20]. Chronic inflammation is now recognized as a main factor in many major disorders, including pulmonary, metabolic and neurological diseases [21,22,23]. Topical inflammatory disorders including dermatitis and psoriasis are common examples where local tissue inflammation disrupts skin integrity, leading to discomfort and increasing health burden [24,25]. Although steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) are effective, they often cause serious side effects and are not universally accessible [25,26]. In this context, medicinal herbs have appeared as promising alternatives, offering anti-inflammatory benefits with fewer adverse effects, greater affordability, and widespread availability [27]. Numerous studies have demonstrated the efficacy of plant-derived phytoconstituents, for instance, flavonoids, alkaloids, and phenolic compounds in modulating inflammatory pathways [28,29,30]. Therefore, exploring plant-based therapies is crucial for advancing safer and more effective management of inflammatory disorders [31].

Transcutol® P (TP) and Capryol® 90 (CP90) are widely used excipients in topical drug delivery systems, known for their ability to enhance the permeation of active pharmaceutical ingredients (APIs) through the skin [32]. TP is a diethylene glycol monoethyl ether with the chemical formula C6H14O3. This clear, low-viscosity liquid acts as a powerful solubilizer, significantly increasing the solubility of both hydrophilic and lipophilic drugs. Its polar nature allows it to disrupt the lipid structure of the stratum corneum, enhancing drug diffusion and facilitating deeper penetration into the skin layers [32,33].

CP90, on the other hand, is a propylene glycol monocaprylate, primarily composed of caprylic acid esters. With a chemical formula of C10H20O3C10H20O3, it serves as a nonionic surfactant that is a solubilizing agent and also enhances skin penetration by modifying the lipid composition of the stratum corneum. Its lipophilic characteristics improve drug solubility and facilitate the transport of APIs across the skin barrier [34].

Together, these excipients demonstrate a synergistic effect in topical formulations, where TP increases drug solubility while CP90 enhances permeation, leading to improved bioavailability and therapeutic efficacy of topical medications [35].

Ointments are semi-solid formulations that play a significant role in topical drug delivery systems, primarily due to their capacity to provide localized treatment while minimizing systemic absorption. These formulations are typically composed of a base that may be oleaginous, absorption-type, or emulsion-based, which facilitates the effective solubilization and penetration of active pharmaceutical ingredients (APIs) through the skin barrier. A notable advantage of ointments lies in their ability to deliver medications directly to specific areas of the skin. This targeted application is particularly advantageous for the treatment of localized conditions, such as dermatological disorders or localized pain. The oily composition of ointments enhances the solubilization of lipophilic drugs, thereby improving their absorption through the stratum corneum, which constitutes a significant barrier to drug entry into the systemic circulation. Furthermore, ointments form a protective layer on the skin that aids in moisture retention, making them particularly suitable for managing dry or irritated skin conditions [3,36,37].

Another important aspect regarding the examination of our selected medicinal plant is that there is no study of the bioactive components of C. erythraea collected in our country. A detailed review published in 2021 collected studies from all countries concerning the phytochemical studies of C. erythraea, and the data published from Romania is very incomplete [10,38]. Among other things, the purpose of our research is to improve the existing bibliography because the number of sites where this plant occurs is very limited. Even though there are few studies on the medicinal plant, its medicinal use is widespread in several countries; in Poland, for example, about 10 tons of the raw materials of C. erythraea are collected each year [39]. The convergence of ethnomedical documentation and contemporary pharmacological evidence underscores the therapeutic relevance of this species within traditional European medicine. Phytochemical analyses have revealed a complex profile dominated by xanthonoids, flavonoids, and phenolic acids, compounds known to mediate a broad spectrum of bioactivities. Such findings substantiate its empirical use as a bitter tonic, diuretic, and anti-inflammatory agent, providing a mechanistic basis for its antioxidant, hepatoprotective, and gastroprotective effects [4,10,40].

The main objectives of the present study were to formulate ointments containing C. erythrea extract with different excipients that have solubilizing and penetration enhancer effects in order to achieve a good diffusion profile with antioxidant and anti-inflammatory effects.

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Materials

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT powder), Dulbecco’s Modified Eagle’s Medium (DMEM), phosphate-buffered saline (PBS), TrypLE™ Express Enzyme, heat-inactivated fetal bovine serum (FBS), L-glutamine, non-essential amino-acid solution, and penicillin–streptomycin were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA). Culturing flasks and 96-well plates were purchased from Thermo-Fisher (Darmstadt, Germany, CAS number: 156499). Cetostearyl alcohol, white petroleum, beeswax, polysorbate 60 and conserving solution were obtained from Hungaropharma Ltd. (Budapest, Hungary). HaCaT cells were supplied from Cell Lines Service (CLS, Heidelberg, Germany). Transcutol® P and Capryol® 90 were a kind gift from Gattefossé (Lyon, France). FRAP assay (P. No.:MAK509), 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid monohydrate (CAS Number: 5995-86-8), Folin and Ciocalteu′s phenol reagent, sodium carbonate decahydrate (CAS Number: 6132-02-1), human IL-1β ELISA Kits were purchased from Sigma-Aldrich (Budapest, Hungary).

Karetka, A.J.; Papp, B.; Lekli, I.; Vlase, A.-M.; Pallag, A.; Vicaș, L.G.; Lestyán, A.-M.; Józsa, L.; Kósa, D.; Pető, Á.; et al. In Vitro and in Vivo Efficacy of Different Ointment Formulations Containing Centaurium erythraea Rafn. Aerial Extract. Pharmaceuticals 2025, 18, 1681. https://doi.org/10.3390/ph18111681